After cleavage of its 21 amino acids signal peptide (Cohen et al., 1996), mature human leptin is secreted as a 146 amino acid protein, with a typical type II interleukin structure, consisting of a bundle of 4 helices (helix 1-4), with an up-up-down-down topology (Zhang et al., 1997). Leptin is secreted into the bloodstream primarily by adipocytes, and blood concentrations of leptin correlate with white adipose tissue mass. Leptin acts as an energy homeostasis hormone, regulating energy expenditure and food intake.
Leptin does so by binding to the leptin receptor in certain areas in the hypothalamus, which leads to phosphorylation of STAT molecules that subsequently migrate to the cell nucleus and induce transcription of different genes.
In addition to its adipostatic function, leptin has many other functions: it can induce proliferation, differentiation and functional activation of hemopoietic cells (Gainsford et al., 1996), and induces angiogenesis (Sierra-Honigmann et al., 1998).
Leptin also interacts with the immune and inflammatory responses (Loffreda et al., 1998).
Leptin levels are acutely increased by inflammatory stimuli and by pro-inflammatory cytokines TNF-α and IL-1 (Grunfeld et al., 1996). Leptin itself regulates the production of several cytokines in vitro, regulates the T helper (Th1/Th2) balance, and can up-regulate inflammatory responses (Loffreda et al., 1998; Faggioni et al., 1998; Lord et al., 1998).
The human leptin receptor is expressed at the cell surface of many different tissues. At least six different splice variants of the human leptin receptor were found at present. The longest isoform of the human leptin receptor consists of 1162 amino acids, with an extracellular region between residues 1 and 840, a transmembrane region between residues 841 and 863 and an intracellular region between residues 864 and 1162.
The extracellular part of the human leptin receptor contains at least 7 structural domains (Fong et al., 1998).
Domain 1 (residue 62-178) and 2 (residue 235-328) have a fibronectin type III fold and together form a cytokine receptor module (CRM), named CRM1.
Domain 3 (residue 329-427) has an Immunoglobulin type fold.
Domain 4 (residue 428-535) and 5 (residue 536-635) also have a fibronectine type III fold and together form a second cytokine receptor module (CRM), named CRM2.
Domains 6 and 7 have a fibronectin type III domain structure.
Like all members of the class I cytokine receptor family, the leptin receptor has no intrinsic kinase activity, and uses a cytoplasmic associated Janus kinase (JAK2 in case of the leptin receptor) for intracellular signaling (Ghilardi et al., 1997). In a generally accepted model, leptin binding leads to formation of a receptor complex, allowing activation of JAK2 by cross-phosphorylation. Activated JAK2 then rapidly phosphorylates several tyrosine residues in the cytosolic domain of the leptin receptor. These phosphorylated tyrosine residues provide docking sites for SH2 containing signaling molecules. In the mouse leptin receptor, tyrosine 1138 serves as a binding site for signal transducer and activator of transcription 3 (STAT3) (Baumann et al., 1996). STAT3 itself is a substrate for JAK2 and dimerizes upon phosphorylation, translocates to the nucleus and modulates transcription of target genes.
The leptin receptor shows the highest sequence similarity with the cytokine receptors of the IL-6 family and with the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor. FSSP (Holm and Sander, 1997) structural similarity searches reveal that leptin shows the highest structural similarity with the cytokines of the IL-6 family and G-CSF, and to a lesser extent with other long chain cytokines, such as the growth hormone and placental lactogen. The crystal structure of the Kaposi's sarcoma-associated herpes virus IL-6 (vIL6, viral IL-6) in a 2:2 complex with the three N-terminal extracellular domains of human gp130 reveals two binding sites, binding site II and binding site III, for interaction between vIL6 and gp130 (Chow et al., 2001). Binding site II, consisting of residues in helices 1 and 3 of vIL6, interacts with the cytokine receptor module (CRM) of gp130. Binding site III in vIL6 consists of residues in the N-terminus of helix 4, in the loop connecting helix 3 and 4 and in the loop connecting helix 1 and 2, and interacts with the Immunoglobulin-like domain of gp130. Corresponding site II and III residues were identified in other members of the IL-6 family of cytokines by site directed mutagenesis: human IL-6, human IL-11, Leukemia inhibitory factor (LIF), oncostatin M (OSM) and Ciliary neurotrophic factor (CNTF) (Kalai et al., 1997; Savino et al., 1993; DiMarco et al., 1996; Hudson et al., 1996; Inoue et al., 1995; Barton et al., 1999; Bravo and Heath, 2000).
IL-6 contains a third binding site, binding site I, for interaction with the IL-6 α receptor. Human IL-6 forms a hexameric 2:2:2 complex with its gp130 and IL-6 α receptor chains: each IL-6 molecule binds two gp130 molecules by its site II and III binding sites, and one IL-6 receptor a subunit (IL-6Rα) by its binding site I (FIG. 1) (Boulanger et al., 2003).
Activation of the leptin receptor by binding of leptin plays a role in several physiological processes. Several variant and mutant forms of leptin have been described, that can be used in different applications. PCT International Patent Publication No. WO02062833 describes modified leptin polypeptides that are substantially non-immunogenic or less immunogenic than any non-modified counterpart when used in vivo. These polypeptides can be administered to humans of therapeutic use. PCT International Patent Publication No. WO9700319 discloses chimeric leptin polypeptides comprising leptin or a mutant or variant thereof fused to a human immunoglobulin domain. These chimeric derivatives have prolonged clearing rates and may be useful in the treatment or prophylaxis of obesity, or diseases and conditions associated with obesity such as atherosclerosis, hypertension and type II diabetes. PCT International Patent Publication No. WO9720933 discloses mutational variants of the mammalian leptin. These molecules can serve as agonist or antagonist of the wild-type leptin; their capacity to induce the signaling pathway upon binding of the receptor varies for the different muteins. PCT International Patent Publication No. WO9812224 describe the use of fragments, derived from leptin, as leptin antagonist, especially for treating type II diabetes.
A need remains for a leptin mutant that is able to bind to the receptor, with a similar or higher affinity as the wild-type leptin, but without remaining signaling activity. Such leptin mutant would be a powerful antagonist and can be used to treat leptin-mediated diseases.